Evaluation of antimicrobial susceptibility tests for Acinetobacter and Pseudomonas species using disks containing a high dose of meropenem

The emergence and dissemination of carbapenem-resistant species of Acinetobacter and Pseudomonas have become a serious health concern. Routine antimicrobial disk susceptibility tests in clinical laboratories cannot distinguish between isolates that are highly carbapenem-resistant and those that are moderately carbapenem-resistant. The present study describes antimicrobial susceptibility tests using disks containing high doses (1000 μg) of meropenem. The diameters of inhibition zones were significantly negatively correlated with the MICs of Pseudomonas and Acinetobacter species for meropenem (R2: 0.93 and 0.91, respectively) and imipenem (R2: 0.75 and 0.84, respectively). Double disk synergy tests using clavulanic acid or sodium mercaptoacetate can detect ESBL or MBL producers. Susceptibility tests using disks containing high doses of meropenem can easily detect highly carbapenem-resistant isolates in a quantitative manner. These disks may be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.


Double disk synergy tests
Double disk synergy tests were performed using disks containing 1000 μg meropenem, together with disks containing the ESBL inhibitor clavulanic acid (40, 80 or 120 μg) 27 , or disks containing the MBL inhibitor sodium mercaptoacetate (3000, 6000 or 12,000 μg) 28 .Bacterial isolates were inoculated onto MH agar plates, and two disks, one containing meropenem and the other containing clavulanic acid or sodium mercaptoacetate, were separately placed on these plates at center-to-center distances of 5, 10 or 20 mm.The plates were incubated at 35 °C for 18 or 24 h, as described above, and the shape of each meropenem inhibition zone was determined.

Microdilution method
The MICs of imipenem, meropenem, ciprofloxacin and amikacin were also determined by a microdilution method, as described by Clinical Standards Laboratory Institute guidelines 7 .Carbapenemase production was detected with the CIMTrisII carbapenem inactivation method (Kohjin Bio, Saitama, Japan) 24 .

Double disk synergy tests
Double disk synergy tests were performed using a P. aeruginosa isolate that produced GES-5.Placement of a disk containing 10 μg clavulanic acid, together with a disk containing 1000 μg meropenem, on an MH agar plate at a distance of 5 or 10 mm showed that clavulanic acid did not alter the shape of the meropenem inhibition zone (Fig. 4).The shape of the meropenem inhibition zone was also unaffected by placement of a disk containing 40 μg clavulanic acid at distances of 5 and 10 mm (Fig. 5A,B).At a distance of 20 mm, the meropenem inhibition zone was slightly enhanced at the front of the disk containing 40 μg clavulanic acid, altering the shape of the meropenem inhibition zone (Fig. 5C).Placement of a disk containing 80 μg clavulanic acid 20 mm from the disk containing 1000 μg meropenem enhanced the meropenem inhibition zone at the front of the clavulanic acid-containing disk (Fig. 6A).This enhancement of the inhibition zone was also observed when a disk containing 120 μg clavulanic acid was placed 20 mm from the disk containing 1000 μg meropenem, although the clavulanic acid disk alone formed an inhibition zone (Fig. 6B).These findings showed that double disk synergy tests of a P. aeruginosa isolate producing GES-24 with clavulanic acid doses of 80 and 120 μg similarly enhanced the inhibition zone formed by 1000 μg meropenem (Fig. 7).
Double disk synergy tests were also performed using an isolate of NDM-1 producing P. aeruginosa.Separate placement of disks containing 1000 μg meropenem and 3000 μg sodium mercaptoacetate on MH agar plates at distances of 5, 10 and 20 mm showed that, at distances of 5 and 10 mm, the meropenem inhibition zone was slightly enhanced at the front of the disks containing sodium mercaptoacetate (Fig. 8A,B), with greater enhancement observed when the disks were at a distance of 20 mm (Fig. 8C).This enhancement of the inhibition zone was also observed following placement of a disk containing 6000 μg sodium mercaptoacetate (Fig. 9A).Moreover, a disk containing 12,000 μg sodium mercaptoacetate alone formed an inhibition zone (Fig. 9B), as did a disk containing 10 μl of 0.5 M EDTA, an inhibitor of metallo-β-lactamase such as NDM-1 (data not shown).

Discussion
The present study found that antimicrobial susceptibility tests using disks containing a high dose of meropenem were able to quantitatively determine the carbapenem resistance of Acinetobacter and Pseudomonas species (Figs. 2, 3).Of the 125 clinical isolates of Pseudomonas species tested, 70 were highly resistant to meropenem, defined as having MICs ≥ 64 μg/ml for meropenem (Table 1), with 69 of these 70 highly carbapenem-resistant isolates of Pseudomonas having inhibition zones of diameter < 32 mm (Table 3).Moreover, all 42 highly carbapenem-resistant isolates of Acinetobacter species had inhibition zones of diameter < 27 mm (Table 3).These findings suggest that, in double disk synergy tests, a disk containing 1000 μg meropenem and a disk containing 80 μg clavulanic acid should be placed 20 mm apart on an MH agar plate to detect bacteria that produce class A carbapenems, whereas a disk containing 1000 μg meropenem and a disk containing 6000 μg sodium mercaptoacetate should be placed 20 mm apart on an MH agar plate to detect bacteria that produce class B metallo-β-lactamases.Although double disk synergy tests were optimized to detect ESBL and MBL producers, these tests failed to detect OXA-23 and KPC-2 producers.Similar results were observed with double disk synergy tests using standard disks that could not detect producers of OXA-type and KPC-type carbapenemases.Table 2. Acinetobacter species isolates, carbapenemases produced by these isolates and their drug susceptibility profiles (MIC values and inhibition zone diameters using a high dose of meropenem disk).a The isolate harbored an intrinsic bla OXA-51-like or bla ADC gene flanked by ISAba1 which is responsible for the production of an intrinsic charmapenemase, OXA-51-like β-lactamase, and an intrinsic cephalosporinase, ADC (AmpC) β-lactamase.isolates that produce all types of carbapenemases, including OXA-type and KPC-type enzymes.These disks may be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.Antimicrobial susceptibility tests using disks containing a high dose of meropenem will be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.Although screening agar containing carbapenems has been shown useful in detecting carbapenem-resistant isolates of Enterobacteriaceae [29][30][31] , screening agar has not been routinely used in bacteriological laboratories because carbapenems in agar are chemically instable, making laboratory storage difficult.

Conclusion
Susceptibility tests using disks containing a high dose of meropenem (1000 μg) can easily detect highly carbapenem-resistant isolates of Acinetobacter and Pseudomonas species in a quantitative manner.Double disk synergy tests using the disk containing meropenem with a disk containing a high dose of clavulanic acid (80 μg) or sodium mercaptoacetate (6000 μg) can detect ESBL and MBL producers, respectively, although these tests failed to detect OXA-23 and KPC-2 producers.

Table 1 .
Pseudomonas species isolates, carbapenemase produced by these isolates and their drug susceptibility profiles (MIC values and inhibition zone diameters using a high dose of meropenem disk).

Table 3 .
Correlations between MIC values and inhibition zone diameters in isolates of Pseudomonas and Acinetobacter species a .a MICs of meropenem (the cutoff value of MIC: 64 µg/ml) were compared with inhibition zone diameters in isolates of Pseudomonas species (the cutoff value: 32 mm) and Acinetobacter species (the cutoff value: 27 mm).